Non-alcoholic fatty liver disease (NAFLD) encompasses a wide range of conditions characterised by the build-up of fat in the liver cells of people who do not drink alcohol excessively. At one end of the scale is the relatively harmless simple fatty liver, or steatosis, that does not cause significant liver damage. If left unattended this condition may progress to more advanced conditions, some of which may be life threatening. Non-alcoholic steatohepatitis (NASH) is a significant development in NAFLD, corresponding to an aggressive condition characterised by swelling and tenderness in the liver. With intense, on-going inflammation a build up of scar tissue (fibrosis) may form, eventually leading to cirrhosis where irregular bumps, known as nodules, replace the smooth liver tissue and the liver becomes harder. The effect of this, together with continued scarring from fibrosis, means that the liver will run out of healthy cells to support normal functions. This can lead to complete liver failure. Most people with a fatty liver are overweight or obese. As more and more people lead inactive lives and carry extra weight around with them, so the number of cases of fatty liver, in particular NASH, is rising. Therefore, there is a need for diagnostic tests that may provide a robust assessment of the presence of NASH or steatosis in a patient.
There is currently no specific laboratory test for NASH, making it extremely difficult to diagnose since even people who go on to develop fibrosis and cirrhosis may undergo liver damage for many years before symptoms become apparent.
NAFLD may be suspected in subjects with one or more components of the metabolic syndrome, especially obesity and type 2 diabetes, and elevated serum aminotransferase levels [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] in the absence of alcohol abuse or other common causes of liver disease. The only widely accepted test for distinguishing NASH from other forms of disease is a liver biopsy. This process involves passing a fine hollow needle through the skin and into the liver, withdrawing a small tissue of sample that is submitted for histological examination. Apart from the obvious discomfort induced by this invasive procedure, assessment is often subjective and prone to sampling error.
Several methods for the detection of NAFLD have been described to date based on measuring physico-chemical properties. For instance, scanning the liver with imaging equipment (MRI) allows the detection of fat deposits (steatosis) in the liver. WO08041128 describes a method for the diagnosis of NASH based on the determination of the electrical impedance of the liver using a pair of electrodes that are placed in contact with the liver using an open abdominal surgical procedure (laparotomy). However, this method requires direct contact of the electrodes with the liver, making it more appropriate for the detection of NASH in explanted livers before they are transplanted into a receptor, and does not allow the distinction between the different stages of NAFLD.
Transient elastography or FibroScan (Castera L, et al., 2006, Hepatology 43:373-374) has been proposed for the non-invasive diagnosis of liver fibrosis. Its main application is to avoid liver biopsy in assessing disease progression in patients with chronic hepatitis C.
Several predictive panels based on the multivariate analysis of well-established clinical and laboratory variables (such as age, body mass index (BMI), ALT, AST, glucose, insulin resistance, albumin) have been proposed as non-invasive markers for the quantitative assessment of fibrosis (FibroTest, NAFLD fibrosis score), steatosis (SteatoTest) and NASH (NashTest) and more recently the ELF test for the assessment of liver fibrosis in patients with NAFLD (Guha I N, et al., 2008, Hepatology 47:455-460).
Other methods are based on the presence of different polymorphisms in genes involved in lipid metabolism. These polymorphisms may be detected in body fluids and, thus, they can be considered as non- or minimally-invasive methods. For instance, WO06117945 describes a method for the diagnosis of NASH by detecting the T94A genetic polymorphism in FABP1 in a biological sample taken from a subject.
Other methods are based on the determination of the expression levels of one or more proteins or metabolites in body fluids. In particular, WO06082522 describes a method for detecting steatosis in a patient by determining the levels of ApoA1, α2-macroglobulin, alanine aminotransferase, gammaglutamyl transpeptidase and triglycerides.
WO08021192 describes a non-invasive method for the diagnosis and monitoring of liver diseases such as NASH and steatosis based on the determinination of levels of fatty acids and eicosanoids in a body fluid of the patient. However, this method is limited to the identification of lipid species and requires complex fractionation steps of the body fluids before the metabolites can be detected.
Lelliot et al (FASEB J., 2005, 19: 1108-1119) describe a method for detecting tamoxifen-induced NASH based on the determination of metabolic profiles in blood using [1H]-NMR. However, this method is performed in samples obtained by liver biopsy and thus, it is a highly invasive method.
WO07136822 describes a method for the detection of NASH from other NAFLD by determining the phosphorylation state of one or more members of the AKT/mTOR/IRS pathway in adipose tissue from a subject. However, this method requires the extraction of an adipose tissue sample from a patient, thus resulting in a minimally invasive method.
Cleary there is a need for non-invasive methods as alternatives to existing diagnosis methods, reducing patient discomfort and hospital-stay costs whilst providing a more robust, standardised assessment.